Methods and kits for cell release

ABSTRACT

Methods and kits of releasing cells are provided. The method comprises the steps of providing cultured cells on a cell culture support comprising a multi layer polyelectrolyte coating immobilized on a substrate, and releasing the cultured cells from the cell culture support by a releasing solution comprising DMSO. The kit comprises a cell culture support and a releasing solution. The releasing solution comprises DMSO.

FIELD

The invention relates to methods for cell release. More particularly,the invention relates to methods for cell release from a polymer-basedcell culture support.

BACKGROUND

Adherent cells have conventionally been grown on glass surfaces or onpolymer substrates. The surfaces for cell culture are often pre-treatedto enhance cell adhesion and proliferation. Matrices for adherent cellsthat allow on-demand cell detachment or cell release, have long beenneeded in biomedical and biological applications.

Cultured cells may be detached or released from cell culture supports bya variety of methods. Commonly used cell release methods comprisemechanical methods (such as scraping), treatment with proteolyticenzymes (such as trypsin), use of calcium chelators (such as EDTA), or acombination of such methods. However, many of these conventional cellrelease methods may cause adverse effects on cultured cells, and maymodify their inherent structure and function. For example, treatment ofcells with trypsin (i.e., trypsinization) is a harsh method, and is notdesirable for delicate cells such as stem cells, due to its potentialeffect on cell phenotype. Moreover, trypsin is typically derived fromanimals, and may contain impurities like co-fractionated proteins orbiological agents (such as viruses or mycoplasma). Impurities of animalorigin may limit the use of released cells for critical applicationssuch as cell therapy. Mechanical methods for releasing cells are laborintensive and are often impractical for industrial-scale cell cultureapplications.

Other non-enzymatic methods include physical methods that useultrasounds or shock waves, which generate bubbles that facilitate celldetachment. Cultured cells from cell culture supports comprisingthermoresponsive polymers like poly-N-isopropylacrylamide (PNIPAAm) maybe released by cooling the cell culture support to a temperature in arange from about 4-20° C.

Efficient cell release is particularly important for high yield inindustrial-scale cell culture processes. Therefore, there is an emergingneed to develop better cell release techniques for fast, efficient celldetachment without affecting cell morphology and cell viability.

BRIEF DESCRIPTION

The invention generally comprises both methods and kits for releasingcells from a cell culture support.

An example of a method for releasing cells from a cell culture supportcomprises providing cultured cells on a cell culture support andreleasing the cultured cells from the cell culture support by adding areleasing solution. The releasing solution comprises dimethyl sulfoxide(DMSO). The cell culture support comprises a multi-layerpolyelectrolyte-based coating on a substrate.

An example of a kit for culturing cells generally comprises a cellculture support, and a releasing solution for cell release. Thereleasing solution comprises DMSO. The cell culture support comprises asubstrate, and a multi-layer polyelectrolyte-based coating on thesubstrate.

Another example of a method for releasing cells comprises the steps ofproviding cultured human mesenchymal stem cells on a cell culturesupport (comprising a multi-layer polyelectrolyte-based coating on asubstrate), and releasing the cultured cells from the cell culturesupport at room temperature by incubating the cells with a releasingsolution. The releasing solution comprises about 0.01% DMSO in PBS.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1A is an image of an uncoated glass slide after addition of areleasing solution comprising DMSO. FIG. 1B is an image of a glass slidewith a multi-layer polyelectrolyte-based coating after releasing cellsusing a releasing solution comprising DMSO.

FIG. 2 represents graphical plots of a WST-1 cell assay, illustratingthe change in optical density of the WST-1 reagent when mixed withreleased cells and measured at 450 nm. The optical density at 450 nmcorrelates with number of cells present in the assay system. Cells werereleased from uncoated or multi-layer polyelectrolyte coated glassslides using a releasing solution comprising DMSO in PBS.

DETAILED DESCRIPTION

To more clearly and concisely describe the subject matter of the claimedinvention, the following definitions are provided for specific terms,which are used in the following description and the appended claims.Throughout the specification, exemplification of specific terms shouldbe considered as non-limiting examples.

A “cell culture support”, as referred to herein, is a support foradhering and culturing cells. The cell culture support may comprise asubstrate. The substrate may be coated or layered with a suitablecoating material for cell adherence and proliferation. Suitable coatingmaterials may include, but are not limited to, polyelectrolytes.

A “substrate”, as referred to herein, is a base or a holder, whichprovides support for a coating. The coated substrate may be used as acell culture support.

A “releasing solution”, as referred to herein, is a solution that helpsto release or detach cells from a cell culture support. The releasingsolution comprises one or more polar aprotic solvents, e.g. dimethylsulfoxide (DMSO). For example, DMSO belongs to a family of polar aproticsolvents, which are water miscible. Other polar aprotic solvents includebut are not limited to: tetrahydrofurane, acetone,N,N-dimethylformamide. Those solvents strongly interact with polymerssuch as polyelectrolytes and are capable to disturb hydrogen bonds aswell as electrostatic interactions, which affect polymer aggregation andcell adhesion. The releasing solution comprises DMSO in phosphatebuffered saline (PBS).

“Polyelectrolytes” as referred to herein, are polymers wherein therepeating units of the polymer bear an electrolyte group. The polymersbecome charged while these groups are dissociated in aqueous solutions(e.g., water). Polyelectrolyte properties are thus similar to bothelectrolytes (salts) and polymers (high molecular weight compounds), andare sometimes called polysalts. The polyelectrolyte solutions areelectrically conductive like salts and are often viscous like polymers.Many biological molecules are polyelectrolytes. Examples ofpolyelectrolytes include but not limited to polypeptides (or proteins),DNA, and polymers.

A high yield of healthy, viable cells is required for applications suchas drug screening and cell therapy. Industrial-scale cell manufacturingunits or cell bioreactors often employ suitable cell culture supportsfor culturing cells. Efficient non-enzymatic methods for releasing cellsfrom the cell culture support can be particularly useful if the culturedcells are to be used in therapeutic applications.

The methods and kits of the invention for releasing cells from a cellculture support are useful for delicate cells, such as stem cells.Efficient release of normal cells or delicate cells may acquire usingthe methods and kits for cell release of the present invention.

One example of the method for releasing cultured cells comprises thesteps of: providing cultured cells on a cell culture support, andreleasing the cultured cells from the cell culture support by adding areleasing solution comprising DMSO. The cell culture support maycomprise multiple layers (multi-layer) of polyelectrolyte immobilized ona substrate.

The cells are released using a releasing solution comprising DMSO. Theconcentration of DMSO (v/v) in the releasing solution may range fromabout 0.01% to 1.0%. In some embodiments, the concentration of DMSO inthe releasing solution may range from about 0.02% to 0.5%, and 0.1% to0.5%. Cell release efficiency may be enhanced by changing theconcentration of DMSO in the releasing solution. The efficiency for cellrelease may increase with increasing concentrations of DMSO. Thereleasing solution may further comprise DMSO in a phosphate bufferedsaline (PBS) wherein pH of the solution is about 7.5.

The release of the cultured cells may be further optimized by incubatingthe cell culture support with the releasing solution at room temperatureor in ranges from about 15° C. to 37° C., about 20° C. to 35° C. orabout 20° C. to 30° C.

The cells may be released from the cell culture support by incubating onthe cell culture support at room temperature for various periods oftime, such as, for about 1 hr to 2 hrs, about 30 min to 1 hr, or about10 min to 30 min. In some embodiments, cell release efficiency may bemodified, by changing incubation time for cell release after addingreleasing solution. For example, cell release efficiency may beincreased with increasing incubation time.

The method for releasing cells comprises the step of providing culturedcells on a cell culture support. The method may include culturing thecells on the cell culture support. The cells may be cultured at avarious temperature ranges from about 20° C. to 37° C., about 30° C. to37° C. and about 35° C. to 37.5° C., depending on, for example, celltype.

The cells may be grown in a culture flask and may be added to the cellculture support for further growth. Cells may be grown on the cellculture support after extraction from a biological source such as, butnot limited to, blood, bone marrow, or tissue section. In some otherembodiments, the cell culture support may be introduced in a spinnerflask, a stacked culture flask, a stirred tank reactor, or any otherin-vitro cell culture system.

The methods may also be used to release fragile cells once they arecultured on the cell culture support. The fragile cells may include, butare not limited to, embryonic stem cells, adult stem cells, inducedpluripotent stem cells, dendritic cells, hematopoietic cells,mesenchymal stromal cells, neural cells, reprogrammed cells, orde-differentiated cells. For example, cultured stem cells are releasedfrom the cell culture support by incubating the cell culture supportwith a releasing solution comprising 0.01% (v/v) of DMSO for a period ofabout 10 min.

Efficient methods for stem cell culture are critical to generate stemcells having high purity in good yield for use in clinical or researchapplications. Culture of stem cells often require specialized techniquessince the stem cells may lose their multipotency or pluripotency or maydifferentiate during cell culture. More over, conventional methods ofreleasing cells, such as mechanical scraping or trypsinization, may notbe suitable for releasing the cultured stem cells. These issues may beaddressed by using one of the non-enzymatic examples of the methods forreleasing cells. For example, mesenchymal stromal cells may besuccessfully cultured and released using the non-enzymatic, DMSO-basedcell release method described herein without any detrimental effects onthe cultured mesenchymal stromal cells.

The cell culture support may be configured as a cell culture bed, a cellcarrier bead, disk or scaffold comprising one or more polymeric layers.Non-limiting examples of substrates include a microcarrier, a membrane,a fiber, a hollow fiber, a capillary, a vessel, a flask, a disc, a bead,a Petri dish, a plate, a woven or non-woven fabric or mesh, a nano-fibermat, a particle, a scaffold or a foam. Examples of substrate materialsinclude, but are not limited to, glass, polymer, metal, ceramic andcombinations thereof.

The cell culture support may comprise a multi-layerpolyelectrolyte-based coating. The multi-layer polyelectrolyte-basedcoating may comprise at least one copolymer layer or at least onehomopolymer layer. In other embodiments, the multi-layerpolyelectrolyte-based coating may comprise at least one copolymer layerand at least one homopolymer layer. The copolymer layers and thehomopolymer layers may also be stacked in the coating in an alternatingarrangement. For example, the cell culture support may comprisealternate blocks of a copolymer layer and/or a homopolymer layer. Themultiple layers of polyelectrolyte-based coating may be fabricated byutilizing a layer-by-layer (LBL) technique. One or more polymer layersmay be arranged one over another by using the LBL technique.

The multi-layer polyelectrolyte-based coating may comprise a copolymerwherein the copolymer may be a block copolymer. The coating may comprisemultiple layers of identical or different block copolymers. Anon-limiting example of a block copolymer is a thermoresponsiveamphiphilic block copolymer (TRABC). The block copolymer may comprisepoly (di (ethylene glycol) methylether methacrylate)-co-poly(acrylicacid). In addition to the block copolymer, the multi-layer coating mayfurther comprise one or more additional polymer layers (e.g. homopolymeror copolymer). Non-limiting examples of homopolymers include poly(L-lysine), poly (allylamine), poly (ethylene imine) and poly(vinylpyrrolidone).

The homopolymer or copolymer in the multi-layer polyelectrolyte coatingmay be responsive to one or more external stimuli such as temperature,pH, or ionic strength. The homopolymer may be responsive to ionicstrength or pH or both. The copolymer may be responsive to temperatureor pH or both.

The multi-layer polyelectrolyte-based coating may be adhered to asubstrate via non-covalent interaction. Attaching the polymer coatingnon-covalently to the substrate offers significant advantages, such as,the flexibility to use the substrate in complex substrate geometries(e.g., flat sheets, beads, cubes, porous foams, fibers and nonwovens).

An embodiment of the kit of the invention, for culturing and releasingcells, may comprise a cell culture support having a substrate withmulti-layer polymer-based coating on the substrate, and a releasingsolution. The kit may further comprise cells (e.g., in a frozencondition) and/or medium for culturing cells. The kit may furthercomprise a protocol for handling, culturing and/or releasing cells fromthe cell culture support. The kit may be packaged along with a manualdescribing the method of using the kit.

Example 1 Preparation of Multi-Layer Polymer Coated Cell Culture Support

Multi-layer polymer coated cell culture supports were prepared asfollows. Poly-L-Lysine (PLL) coated glass slides (Polysciences Inc.)were diced into 8×8 mm squares. The slides were washed with de-ionized(DI) water and with absolute ethanol followed by air-drying at roomtemperature. The dry slides were immersed into 0.1% solution ofpoly(di(ethyleneglycol)methylether methacrylate)-co-poly(acrylic acid)(PDEGMEMA-co-PAA) in DI water at 37° C. for 60 minutes. The slides werethen washed with DI water and were incubated with 0.1% solution (w/v) ofPoly-L-Lysine (Aldrich) for 60 minutes at 37° C. The 0.1% solution ofpoly-L-Lysine (PLL) contained a small amount (about 0.1 mol %) ofpoly-L-lysine functionalized with fluorescein 5-isothiocyanate(FITC-PLL) (FITC from Sigma) fluorescent probe. Introduction of afluorescent probe enables the measurement of the fluorescence intensityof polymer layers to determine proper formation of multi-layer coatings.The slides were washed with warm (37° C.) DI water and were thenincubated with 0.1% solution (w/v) of poly(di(ethyleneglycol)methylethermethacrylate)-co-poly(acrylic acid) for 60 min at 37° C. Slides werewashed with DI water and air-dried. The amount of deposited PLL wasdetermined by measuring the intensity of FITC florescence on a Typhoonfluorescence imager.

Quantitative analysis of the measured fluorescence confirmed theformation of PLL/PDEGMEMA-co-PAA five-layer coating (FITC bound). Theformation of PLL/PDEGMEMA-co-PAA coating on the substrate wasadditionally confirmed by time of flight secondary ion mass spectrometry(ToF SIMS) analysis (data not shown). The ToF SIMS spectra showed agradual decrease in the peak height and peak position for the observednegative SiO₂ ions. The LBL coating of samples having fivePLL/PDEGMEMA-co-PAA layers was thick enough that SiO₂ ions were almostundetectable (data not shown) with respect to an uncoated glass slide.

Example 2 Cell Culture on the Multi-Layer Cell Culture Support andSubsequent Cell Release

The cell culture support was used to culture human mesenchymal stemcells (hMSC, ATCC). These cells were first cultured on polystyrenesurfaces using the Mesenchymal Stem Cell Growth Medium (PT-3001, Lonza).To culture cells, about 10⁵ human mesenchymal stem cells (primaryculture) were seeded on the surface of the cell culture support(uncoated and/or coated glass slides) and incubated at 37° C., in ahumidified atmosphere of 5% CO₂.

Cells were allowed to proliferate for one week on uncoated and/or onpolymer-coated glass surfaces in the cell culture incubator. Cellexpansion was monitored by bright field microscopy and medium wasexchanged every 3-4 days. Cell-release was tested by adding a releasingsolution comprising 0.01% (v/v) DMSO in PBS. Media was removed from thecell culture support (uncoated or coated glass slides) when the cellswere ˜100% confluent. The cells were rinsed with PBS followed byaddition of enough releasing solution to cover the growth surface withattached cells. The cell culture support with releasing solution wasincubated for 30 minutes at a room temperature. The cells were observedin an inverted microscope until the cell layer was dispersed.Subsequently, released cells were removed from the cell culture supportby pipetting out from the cell culture support. FIG. 1A shows an imageof an uncoated cell culture support (2) after the attempt of releasingcultured human mesenchymal stem cells by using a releasing solutioncomprising 0.01% (v/v) DMSO. FIG. 1B shows an image of multi layerPLL/PDEGMEMA-co-PAA (five layers) coated cell culture support (4) afterthe release of human mesenchymal stem cells (6) by using a releasingsolution comprising 0.01% (v/v) DMSO.

Example 3 Quantitative Estimation of Released Cells

The quantitative measurement of cell release is presented in FIG. 2 bymeasuring the optical density of the cell proliferation assay solutionat 450 nm using WST-1 assay reagent. Released cells were pelleted downby centrifuging the cell suspension at 500 g for 5 min. The pelletedcells were re-suspended in 200 μl growth medium and were transferred in96 well plate. The plate was spun down at 500 g for 5 min. 150 μl freshgrowth medium and 15 μl of WST-1 cell assay reagent were added to eachwell. Cells with assay reagent were incubated at 37° C. for 2.5 hr. Thisexperiment was performed in replicates of 3 different sets. Cell releasefrom uncoated glass slides (bar 8), and five layers ofPLL/PDEGMEMA-co-PAA coated glass slides (bar 10) are shown in FIG. 2.The data shows efficient cell release for the five layers ofPLL/PDEGMEMA-co-PAA coated glass slides (10) compared to uncoatedsurface of the glass slide (8). An uncoated glass slide served as acontrol. The difference between the signal for released cells fromuncoated glass slide and signal for released cells from five layerPLL/PDEGMEMA-co-PAA coated glass slide gives a measure of the releaseefficiency, which as indicated by FIG. 2 is clearly efficient formulti-layer polymer coated cell culture support.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the scope of the invention.

1. A method of releasing cells, comprising: a) providing cultured cellson a cell culture support, wherein the cell culture support comprises amulti layer polyelectrolyte coating immobilized on a substrate; and b)releasing the cultured cells from the cell culture support by adding areleasing solution comprising DMSO.
 2. The method of claim 1, whereinthe cultured cells comprise mesenchymal stromal cells.
 3. The method ofclaim 1, wherein the cultured cells comprise human mesenchymal stromalcells.
 4. The method of claim 1, wherein concentration of DMSO in thereleasing solution ranges from about 0.01% to 1.0%.
 5. The method ofclaim 1, wherein concentration of DMSO in the releasing solution rangesfrom about 0.02% to 0.5%.
 6. The method of claim 1, whereinconcentration of DMSO in the releasing solution ranges from about 0.1%to 0.5%.
 7. The method of claim 1, wherein the cultured cells arereleased from the cell culture support by incubating the cell culturesupport at room temperature.
 8. The method of claim 7, wherein theincubation is performed for about 10 to 30 minutes.
 9. The method ofclaim 1, wherein the multi layer polyelectrolyte coating comprises atleast one homopolymer layer.
 10. The method of claim 1, wherein themulti layer polymer coating comprises at least one copolymer layer. 11.The method of claim 1, wherein the multi layer polymer coating comprisesat least one homopolymer layer and at least one copolymer layer.
 12. Themethod of claim 1, wherein the multi layer polymer coating comprises analternating arrangement of a copolymer layer and a homopolymer layer.13. The method of claim 1, wherein the multi layer polymer coatingcomprises at least one layer of temperature responsive polymer
 14. Themethod of claim 1, wherein the multi layer polymer coating comprises atleast one layer of poly-(di(ethyleneglycol)methylethermethacrylate)-co-polyacrylic acid.
 15. The method of releasing cells ofclaim 1, wherein the multi layer polyelectrolyte coating comprises atleast one layer of poly-L-lysine.
 16. A kit for culturing cellscomprising: a cell culture support; wherein the cell culture supportcomprises a substrate, and a multi layer polyelectrolyte coatingimmobilized on the substrate; and a releasing solution for cell release,wherein the releasing solution comprises DMSO.
 17. The kit of claim 16,further comprising a medium for culturing cells.
 18. The kit of claim16, wherein the multi layer polyelectrolyte coating comprises at leastone layer of temperature responsive polymer.
 19. The kit of claim 16,wherein the multi layer polyelectrolyte coating comprises at least onelayer of poly-(di(ethyleneglycol)methylethermethacrylate)-co-polyacrylic acid.
 20. The kit of claim 16, wherein themulti layer polyelectrolyte coating comprises at least one layer of ahomopolymer.
 21. The kit of claim 16, wherein the multi layerpolyelectrolyte coating comprises at least one layer of poly-L-lysine.22. A method of releasing human mesenchymal stem cells, comprising: a)providing cultured human mesenchymal stem cells on a cell culturesupport, wherein the cell culture support comprises a multi layerpolyelectrolyte coating immobilized on a substrate; and b) releasing thecells from the cell culture support at room temperature, by incubatingwith a releasing solution comprising about 0.1% DMSO.